Forensic DNA analysis of sexual assault evidence often involves analysis of DNA from sperm cells and DNA from other cells such as epithelial cells. Sperm cells are normally obtained from a rape victim by rubbing a swab against a mucous membrane. The samples obtained from victims often contain a mixture of sperm and epithelial cells. Because the epithelial cells may outnumber sperm cells in the sample by at least an order of magnitude, the former can cause contamination of sperm cell DNA, when sperm DNA is purified. Consequently, it is often desirable to separate, as cleanly as possible, the sperm cells and epithelial cells, or the sperm DNA and the epithelial DNA, prior to analysis. Separation and isolation of DNA from sperm and epithelial cells are essential steps in identifying an assailant from a forensic specimen, and in associating the assailant with the victim.
The standard method for purifying sperm from swabs is based on differential extraction. Separation of the sperm DNA from the victim's DNA removes ambiguity, facilitates DNA analysis and allows for easier interpretation of the assailant's DNA profile in a rape case. Although differential extraction is commonly used to separate sperm and epithelial cells, the standard protocol is time consuming and laborious, and entails epithelial cell lysis prior to sperm cell lysis.
Typically, cells are first resuspended from a forensic specimen, followed by selective digestion of the victim's epithelial cells with a solution containing Proteinase K and SDS (sodium dodecyl sulfate). The intact sperm are separated from the solubilized, contaminating DNA and epithelial cell debris by centrifugation, careful removal of supernatant, and extensive washing of the sperm pellet (see e.g., Giusti et al., J. Forensic Sci., 31:409-417, 1986; Gill et al. Nature 318:577-579, 1985; Wiegand et al., Int J. Legal Med., 104:359-360, 1992; and Yoshida et al., Forensic Sci. Int., 72:25-33, 1995). Unfortunately, the processes of centrifugation and careful removal of supernatant are difficult to automate and can cause the loss of sperm DNA due to multiple sample handling steps.
In one example of this procedure, Gill et al. (supra) describe a process for isolating sperm DNA from vaginal swabs taken from sexual assault victims. These swabs contain sperm and also a large excess of the victim's epithelial cells. The epithelial cells and the DNA contained in these cells are removed by preferential lysis (i.e., by incubation of the cell mixture in a buffer solution containing SDS, and Proteinase K). Sperm nuclei are impervious to this treatment because they have disulfide bond cross-linked thiol-rich proteins, while other cell types are digested and the corresponding DNA is solubilized. After preferential lysis, the samples are centrifuged to separate the sperm nuclei from the victim's solubilized DNA. The supernatant containing the victim's DNA is removed and the sperm pellet is washed repeatedly. The sperm nuclei are subsequently lysed by treatment with a buffer solution containing SDS, proteinase K and DTT (dithiothreitol), and the lysate separated from the contaminating cells by centrifugation.
Wiegand et al. (supra) attempted to improve on the method of Gill et al. for samples having low sperm counts by using mild lysis conditions and by avoiding the washing steps.
A number of proposals for separating sperm cells from epithelial cells are based on filtration. Chen et al. (J Forensic Science 43:114-118, 1998) and Garvin (PCT/US01/01835) separate the sperm from the epithelial cells before differential lysis by gravitational or mild vacuum filtration or by use of a filter material that can withstand strong vacuum or centrifugal forces without having the pores increase in size. DNA is then isolated from the sperm collected in the filtrate.
Differential extraction, specifically for sperm cell analysis, has been carried out by first lysing epithelial cells, before extracting the DNA from sperm. In order to reduce the time for forensic analysis, it is desirable to selectively extract the DNA from the sperm cells, initially. In addition, because the selective epithelial cell lysis conditions also cause some sperm lysis, lysing epithelial cells before the sperm cell fraction reduces the number of sperm cells present in the sample. The additional wash steps that are required to remove epithelial DNA also contribute to the loss of sperm cells.
Accurate sperm analysis can be impeded by the lysing or presence of epithelial cells, or other contaminating cell types, such as leukocytes. DNA present in the forensic sample, from damaged nonsperm cells, can contaminate the cells (most likely by binding to the surface of sperm cells), which can inhibit accurate identification of a perpetrator, especially when the number of recovered sperm cells is low.
The present inventor discovered that although techniques exist for differential extraction of one type of nucleic acid (from an individual cell type) from a heterogeneous cell population, residual contaminating nucleic acid is still a problem that warrants more precise extraction methodologies. One application where the need exists for highly specific differential extraction is forensic, specifically sexual assault analysis.
Because of their fragile nature, epithelial and other cells, e.g., leukocytes, in a sexual assault sample often break and lyse during handling, sample processing or storage. This residual DNA, if not removed, may contaminate male DNA during selective sperm lysis, especially when the number of sperm cells is low. In addition, epithelial and sperm cell surfaces are highly functionalized, with molecules (e.g., glycoproteins) that bind nucleic acid through ionic interactions, as well as other non-covalent methods. These cell surface proteins have been found to associate with extracellular nucleic acid present in a sample.